Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations1,2. Even though some unfolded and partially folded proteins are now known to possess biological function3 or to be involved in aggregation phenomena associated with disease states1,4, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a ‘Trp-cage’ miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR6 pulse-labelling experiments that involve rapid in situ protein refolding7,8. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.
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We thank I. Kuprov for the synthesis of 9-fluorenylmethoxycarbonyl-O-t-butyl-3-fluoro-l-Tyr and for NMR experiments on the 19F-Tyr-TC5b variant; T. Nagashima, C. J. V. Jones and H. Paisley for help with the conceptual design, building and testing of the rapid mixing injector; R. Gerber for assistance in acquiring the NMR spectra; A. L. Davis for discussions and for providing spectrometer time for the diffusion measurements; L. J. Smith, C. Redfield, A. E. Mark and D. A. C. Beck for discussions; and S. Min for assistance in figure preparation. K.H.M. also thanks M. Nilges, R. Wade and the EMBO Practical Course on Biomolecular Simulation. We are indebted to C. M. Dobson for continued encouragement in the application of photo-CIDNP to protein folding problems. This work was supported by the BBSRC (K.H.M., L.T.K., and P.J.H.), the Studienstiftung des deutschen Volkes (L.T.K.), the Deutsche Forschungsgemeinschaft (M.G.), and the US National Institutes of Health (N.H.A. and J.C.L.).
Author Contributions K.H.M. and I.J.D. built the in situ rapid mixing injector. K.H.M., M.G., I.J.D. and P.J.H. designed the experiments. K.H.M., L.T.K., M.G. and I.J.D. performed the experiments. J.C.L. and N.H.A. contributed the TC5b sample. M.G. developed the mathematical methods for obtaining NOE contact distances. K.H.M., L.T.K., M.G., I.J.D., N.H.A. and P.J.H. analysed the data. K.H.M., M.G., N.H.A. and P.J.H. wrote the paper. All authors discussed the results and commented on the manuscript.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Mok, K., Kuhn, L., Goez, M. et al. A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein. Nature 447, 106–109 (2007). https://doi.org/10.1038/nature05728
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